Summary of Work: The broad aim of this project is to understand at the cellular and molecular level the mechanisms involved in regulation of calcium entry across the plasma membrane in non-excitable cells. This is a process that is ubiquitous to non-excitable cells, occurs in many excitable cell types and which is known to play an important role in the control of a large variety of cell processes, including secretion, contraction, motility, growth, differentiation and apoptosis. This is a signalling process vulnerable to environmental intervention by chemical and physical agents (such as EMF). Capacitative calcium entry is believed to involve an unknown signal generated in the endoplasmic reticulum when Ca2+ is released by IP3 and which then diffuses to the plasma membrane to activate calcium channels. The nature of this signal and the nature of the channels involved are prime areas of investigation. We utilize as a model for studying this process the actions of a tumor- promoting plant product, thapsigargin. Thapsigargin acts by inhibiting endoplasmic reticulum Ca2+ pumps, thereby depleting intracellular Ca2+ stores in a passive manner. We are using molecular techniques of reverse transcriptase - PCR and cloning to isolate and study the candidate capacitative calcium entry channel molecules. To date, as many as 6 members of the Htrp family of ion channels have been identified in human cells. We are expressing Htrp-1 and Htrp-3 in cell types in which they are normally expressed and in cell types which do not express these channel proteins. In addition, we are working to obtain full-length clones of other members of the Htrp family. We also consider the type 3 IP3 receptor to be a candidate for the plasma membrane capacitative calcium entry channel, and will attempt to utilize molecular techniques to alter its level of expression and function in cell types in which it is likely to play a role. We are hopeful that by achieving a better understanding of the molecular and cellular modes of regulation of this important signalling pathway, we can learn how capacitative calcium entry is altered by environmental factors and by disease states.